Recent advances in nano-enabled agriculture for improving plant performance

Honghong Wu, Zhaohu Li

a MOA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China

b College of Agronomy and Biotechnology, China Agricultural University, Beijing 100083, China

c Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Shenzhen 518120, Guangdong, China

d Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, Guangdong, China

Keywords:Nano-enabled agriculture Nanosensors Mechanisms Photosynthesis Signaling molecules Stress tolerance

ABSTRACT Nano-enabled agriculture is an emerging hot topic.To facilitate the development of nano-enabled agriculture, reviews addressing or discussing the applications, knowledge gap, future research needs, and possible new research field of plant nanobiotechnology in agricultural production are encouraged.Here we review the following topics in plant nanobiotechnology for agriculture:1)improving stress tolerance,2)stress sensing and early detection,3)targeted delivery and controlled release of agrochemicals,4)transgenic events in non-model crop species,and 5)seed nanopriming.We discuss the knowledge gaps in these topics.Besides the use of nanomaterials for harvesting more electrons to improve photosynthetic performance, they could be used to convert nIR and UV to visible light to expand the light spectrum for photosynthesis.We discuss this approach to maintaining plant photosynthesis under light-insufficient conditions.Our aim in this review is to aid researchers to learn quickly how to use plant nanobiotechnology for improving agricultural production.

1.Introduction

Nanomaterials are defined as materials having one dimension less than 100 nm.In contrast to their conventional counterparts,a key advantage that nanomaterials possess is a high surface-tovolume ratio due to their small size.They have unique physicochemical properties:small surface area, atypical surface structure,and increased reactivity due to their small size, surface structure,chemical composition, stability, shape, and agglomeration of nanoparticles [1].Besides its unique physicochemical properties,nanomaterials are readily amenable to surface conjugation and thus can be developed as versatile platforms with broad applications in plant science[2,3].Nanotechnology has shown high potential in agriculture:it can 1) improve plant stress tolerance via ROS scavenging by nanozymes (nanomaterials which can mimic antioxidant enzyme activities) [4,5].Using cerium oxide nanoparticles, researchers increased plant tolerance to salinity [6-8],drought [9], heat [10], and cold [10].2) enable controlled release and targeted delivery of agrochemicals[11,12],3)be used to create nanosensors for early stress detection in plants[13,14].Use of carbon nanotubes enabled the sensing of H2O2[15,16], NO [15,17],and Ca2+[18].4) act as a platform for delivery of DNA or RNA to allow genetic engineering in non-model plant species [19-21],and 5) increase plant tolerance to stress via priming seeds with nanomaterials (nanopriming) [22-24].More applications of nanotechnology tools in agriculture will be introduced in the following sections.

To feed a population of over 9 billion in 2050 [25], agricultural production must increase by 60% from the 2005-2007 level [26].Besides efforts in breeding programs,farm management,and cultivation practices,new approaches such as nano-enabled agriculture have potential for meeting the predicted food shortage.Nanoenabled agriculture, an emerging field [27,28], has potential not only to improve plant tolerance of biotic and abiotic stresses, but to improve plant breeding and agriculture.Thus, besides addressing food shortage in 2050,plant nanobiotechnology could also play an important role in sustainable agriculture.

Previous reviews of nano-enabled agriculture focused more on nanoparticles [29,30], nanotoxicity [31], and nanosensors [14],and less on the potential and flexibility of plant nanobiotechnology in agricultural production[32,33].With proper control and design of nanomaterials, their biosafety issue could be largely addressed[34,35].Reviews of nanotoxicity in plants are available [36-40].Here we focus on the use of nanomaterials in sustainable agriculture.Improvement of plant stress tolerance, stress sensing and early detection, targeted delivery and controlled release of agrochemicals, transgenic events in non-model crop species, and seed nanopriming are reviewed.Knowledge gaps and future research needs are discussed.Potential new projects for nano-enabled agriculture,such as nano-enabled delivery of the CRISPR-Cas system in plants, nanomaterials for converting nIR and UV to visible light to expand the light spectrum available for photosynthesis, and using nanomaterials to harvest electrons to enhance plant photosynthesis, are proposed.Our aim in this brief review is to encourage researchers, especially those outside the field of plant nanobiotechnology, to engage with nano-enabled agriculture.Fig.1 illustrates how plant nanobiotechnology can contribute to sustainable agriculture.

2.Nano-enabled plant stress tolerance:A promising approach for increasing agricultural production

2.1.Nano-enabled plant stress tolerance:Nano vs.Bulk

Fig.1.Use of plant nanobiotechnology for agriculture.Besides improving the efficacy of agrochemicals,a plant nanobiotechnology approach has also shown high potential for targeted delivery and controlled release of agrochemicals.It can enable transgenic events in plants,especially in non-model crops.It can be used to improve stress tolerance,including by stress sensing and early detection.Seed nano-priming may improve crop production.

Comparisons between nano and bulk materials in plant studies and agricultural applications are available:a comparison of nanopesticides and nanofertilizers with their conventional analogs suggested a median gain in efficacy of nanoproducts relative to conventional products of 20%-30% [41].Here we consider why nanoproducts enable higher plant stress tolerance than their conventional counterparts.Owing to changed physical and chemical properties at the nanoscale level, nanomaterials have properties such as catalytic ROS (reactive oxygen species) scavenging ability and self-fluorescence that its bulk or commercial counterparts lack.CeO2nanoparticles are potent ROS scavengers,and are widely used or studied in industry, medical research,and plant science[42].In contrast to bulk cerium oxide, CeO2nanoparticles have a large number of surface oxygen vacancies that alternate between two oxidation states (Ce3+and Ce4+) to confer potent ROS scavenging ability [43,44].They catalytically scavenge ROS; the Ce3+dangling bonds effectively scavenge ROS while nanoscale lattice strains promote the regeneration of surface oxygen vacancies via redox cycling reactions [45].The Mn3O4nanoparticle is a newly developed nanozyme with ROS scavenging ability.In contrast to bulk Mn3O4, a Mn3O4nanozyme showed strongin vivoROS scavenging ability attributed to a 1:2 ratio of two oxidation states, Mn2+and Mn3+[46].Both nanoparticles increased salinity stress tolerance in crops.Cerium oxide nanoparticles (200 mg L-1, size 55.6 nm,zeta potential or surface charge -51.8 mV, soil application)increased salt tolerance inBrassica napus,which under salt stress showed a 48% increase of fresh weight relative to a control [8].In two other studies, polyacrylic acid coated cerium oxide nanoparticles increased salt tolerance inArabidopsis(50 mg L-1,10.0 nm, -17.0 mV, foliar spray) [7] and cotton (100 mg L-1,8.0 nm,-15.3 mV,foliar spray)[6],with plants under salinity stress showing respectively 18%and 40%increases in biomass relative to no-nanoparticle controls.The mechanism was proposed[6,7]to be nanoceria scavenging of ROS and thus modulating the activities of ion channels and transporters to enable better mesophyll K+retention [downregulation of relative expression of KOR (K+outward rectifying channel for K+loss)gene]and shoot Na+exclusion ability[upregulation of relative expression of HKT1(high affinity K+transporter for Na+exclusion) gene].Foliar delivery of Mn3O4nanoparticles (1 mg plant-1, 226.4 nm, -7.7 mV, 7 days) increased salt tolerance in cucumber,which showed under salt stress a 19%increase in total biomass relative to a control [47].Cerium oxide nanoparticles also increased plant resistance to drought[sorghum;CeO2nanoparticles:10 mg L-1, size 15 nm by atomic force microscopy, zeta potential not reported; foliar spray], UV, high light and temperature (Arabidopsis thaliana; CeO2nanoparticles:50 mg L-1, 10.3 nm, -16.9 mV; leaf infiltration) stresses [9,10].

Besides above-mentioned CeO2and Mn3O4nanoparticles,another good example for the comparison between nanoproducts vs.conventional products is carbon-based nanomaterials.Carbon quantum dots(QD)have facile synthesis,high stability,high water solubility, high biocompatibility, strong photoluminescence, tunable surface functionalities, and low toxicity, for potential agricultural applications.Carbon nanodots [50 mg L-1, size 3.9 nm by transmission electron microscopy (TEM), -16.9 mV, leaf infiltration] increased the drought stress resistance of peanut plants[48], although the mechanism was unclear.Multi-walled carbon nanotubes increased salinity stress tolerance inBrassica napus[20 mg L-1, TEM size 6-12 nm (outer diameter), zeta potential not reported, addition into Murashige and Skoog medium] [49]and broccoli (10 mg L-1, TEM size 6-9 nm (outer diameter), zeta potential not reported, hydroponic application) [50] by promoting the production of the gas signaling molecule nitric oxide and aquaporin transduction, respectively.Silicon nanoparticles could be another candidate for increasing plant stress tolerance.Ghorbanpour et al.[51] found that barley plants treated with silicon nanoparticles (125 mg L-1, TEM size 20-30 nm, zeta potential not reported, soil application) showed greater recovery from drought stress and also higher chlorophyll content (up to 17%)and shoot biomass(up to 27%)than a control.Other nanomaterials,such as Fe nanoparticles [52], ZnO nanoparticles [53], and TiO2nanoparticles [54] also improved plant tolerance to abiotic stresses.ZnO nanoparticles (100 mg L-1, 37.7 nm, 14 mV, soil application) improved drought tolerance in maize plants, which showed under drought a 75% increase of proline and 18% decrease of H2O2relative to a control[53].This result was attributed to promotion of melatonin synthesis and an activated antioxidant enzyme system represented by the upregulated gene expression ofFe/Mn SOD,Cu/Zn SOD, andCAT,reducing oxidative damage to mitochondria and chloroplasts [53].

Besides abiotic stresses, biotic stresses such as those from pathogens and insects also result in crop yield and quality penalties.Although pesticides increase agricultural production,growing resistance in fungi and pests and concerns for their effects on human health and the environment [55] call for more efficient and environment-friendly approaches to protecting crops from biotic stress.Nanobiotechnology could assist in developing efficient and green pesticides, such as insecticides [56], fungicides[57], and herbicides [58].A 27% increase of larval mortality was observed inSpodoptera litura(Lepidoptera:Noctuidae) fed on a composite of ZnO nanoparticles with thiamethoxam [ZnO nanoparticles (TEM size 30 nm, zeta potential not reported, leaf dipping with nanoparticles for 10 min), thiamethoxam (10-90 mg L-1)] -impregnated leaves in comparison with the thiamethoxam treatment[56].Many nanomaterials have been tested for positive effects on plant biotic stress tolerance, of which Agand Cu-based nanomaterials are the two most widely studied.Ag-based nanoparticles have the potential not only to suppress disease[59-62],but to inhibit nematodes[63].Similar functions were also found in Cu-based nanomaterials, showing disease suppression [64-67] and pest activity inhibition [68].Nanosheets of Cu3(PO4)2·3H2O (10 mg L-1, 151 nm, zeta potential not reported,foliar application) significantly reduced fungal disease (Fusarium oxysporumf.sp.niveum) in watermelon as measured by a 58%decrease in disease progress, whereas commercial CuO nanoparticles (aggregated in solution, 12 m2g-1surface area) significantly reduced disease (50.6%) only at 1000 mg L-1[67].The higher efficacy of Cu3(PO4)2·3H2O than that of the commercial CuO nanoparticles is attributed to its reduced size,unique structure(sheets)and more rapid initial release of copper ions[67].Other nanomaterials such as carbon nanotubes[69],carbon dots[70],Si-based nanoparticles [71], MgO nanoparticles [72-74], TiO2nanoparticles [75],and CeO2nanoparticles [76] also showed the ability to suppress plant diseases.More detailed reviews of this subject are available[29,35,77].

Plant diseases, insect pests and weeds cause about U.S.$2000 billion in annual agricultural crop losses globally[78],and the cost of fungicide application for pathogen control exceeds$600 million in the United States[79].Pyricularia oryzaecauses rice blast,which results in severe yield reduction (up to 80% in Thailand [80]) in rice;however,the pathogen can rapidly mutate to attack resistant rice lines and can rapidly develop resistance to the fungicides available in the market [80,81].Using nanoproducts to suppress rice blast disease might be an alternative approach.

Water shortage is one of the main constraints to agricultural production, especially in semi-arid areas.Using environmentfriendly nanoproducts to increase crop drought tolerance could allow maintaining or improveing agricultural profit in waterrestricted areas.Nano-enabled plant drought tolerance has been demonstrated in many plant species[9,82,83].As previously mentioned, cerium oxide nanoparticles (nanoceria) improved drought tolerance in sorghum [9].Seed priming with polyacrylic acidcoated nanoceria improved cotton salt tolerance [84].The current cost (quoted by Sigma Aldrich, Saint Louis, MO, USA) of making polyacrylic acid-coated cerium oxide nanoparticles for nanopriming (seed priming with nanomaterials) cotton seed for sowing a 1-ha area is less than $30 and that for a foliar spray of a 1-ha area is ~$100 (personal communications to Prof.Juan Pablo Giraldo at University of California, Riverside).Scaling up production would reduce the chemical cost.Other production costs such as labor,equipment, costs of water, gas and electricity, and rent would add to the cost of a commercial product.

2.2.Nanomaterials without heavy metals and with high dispersibility:Promising candidates for nano-enabled agriculture

Although nano-enabled plant stress tolerance is promising,nanoparticles containing heavy metals,such as quantum dots with Cd2+[85],cerium oxide nanoparticles,and Ag nanoparticles,might raise concerns about biosafety and environmental damage.Although cerium is the most abundant rare-earth element in soil(with about the same abundance as copper) [86], use of cerium oxide nanoparticles to improve plant stress tolerance faces biosafety concerns, given that cerium is a heavy metal.Another concern is the possible aggregation of nanoparticles on plants,which might impair plant stress response[87].Compared to dispersed nanoparticles, aggregated nanoparticles display greatly decreased surface area and reduced cellular uptake, often resulting in toxicity [88].Thus, use of appropriate nanomaterials is necessary for the development of nano-enabled agriculture.One way of increasing the stability of nanomaterials is surface conjugation to reduce the possible leakage of heavy metal elements [89].In a study measuring the intracellular degradation of gold nanoparticles of 4-22 nm in size, the smallest degraded most rapidly [90].Improving the dispersion quality of nanomaterials to avoid aggregation during application [91] would favor uniform biological activity and the adoption of nanomaterials for agricultural practice.Use of copper to interact with chitosan improved the dispersibility of a Cu-chitosan nanomaterial (demonstrated by the induced redistribution of vibrational frequencies recorded by Fourier transform infrared·spectroscopy) in comparison with its bulk counterparts [92].This Cu-chitosan nanomaterial (374 nm, 23 mV, 0.12% concentration,foliar spray) not only increased seedling length (18%) and fresh weight(16%)of tomato relative to a control,but inhibited mycelial growth (71% and 74% inhibition ofA.solaniandF.oxysporum,respectively) and spore germination (61% and 83% inhibition ofA.solaniandF.oxysporum, respectively) of pathogenic fungi [92].

Another strategy to make environment-friendly nanomaterials for agriculture is to explore nanomaterials without heavy metals and with high dispersibility.These materials should be preferred for nano-enabled sustainable agriculture.Testing nanomaterials based on the nutrient elements required by plants shows promise.For example, manganese is an essential micronutrient element for plants, and manganese-based fertilizer is also used in agricultural production.As a newly developed nanozyme,Mn3O4nanoparticles show ROS scavenging ability.Lu et al.[47] reported that Mn3O4nanoparticles increased cucumber salt resistance, suggesting the potential of using Mn3O4nanoparticles to improve crop stress tolerance.

3.Nano-enabled stress sensing and early detection:A strategy for smart agriculture

3.1.Nanosensors for stress sensing

As sessile organisms,crop plants have evolved complex mechanisms to survive stress conditions.Stress sensing and signaling are among these mechanisms·H2O2is a plant signaling molecule [93-95].Besides H2O2, Ca2+, sugars (such as glucose and sucrose), gas molecules (such as nitric oxide, carbon monoxide, and hydrogen sulfide), plant hormones (such as abscisic acid, jasmonic acid,methyl salicylate, and ethylene), and volatile organic compounds(such as isoprenes)are also known as signaling molecules involved in plant stress responses.Ca2+signaling events are involved in signal transduction from root to shoot in plants under salinity stress[96].The profiles of these signaling molecules can be varied under different stresses.Ca2+signatures in plants under salinity and osmotic stress show different peaks [97].However, to date, the non-invasive real-time detection of these signaling molecules such as Ca2+[98],H2O2[99,100],glucose[101]and sucrose[102],is still largely limited in model plant species.Plant nanobiotechnology has been shown to be an efficient approach to monitoring signaling molecules in non-model plant species, such as the ratiometric quantum dot sensor for glucose[85],hemin-complexed DNA aptamer coated single-walled carbon nanotubes (SWCNT) for H2O2[16],AT15-coated carbon nanotubes for NO[15,17,103],and nanoneedle transistor-based sensors for Ca2+[18].Using TGA (thioglycolic acid)-QD (10.2 nm, leaf infiltration, as internal reference because it does not react with glucose) and BA (boronic acid)-QD(11.3 nm, leaf infiltration,as sensor since it aggregates specifically with glucose and thus shows fluorescence quenching), the change of glucose level in leaf (more than 50% BA-QD fluorescence quenching after 60 min incubation of glucose with a QDinfiltrated leaf) was monitored inArabidopsisplants via fluorescence tracking of QDs, showing a 500 μmol L-1detection limit[85].Nanosensors for monitoring temperature [104], humidity[105] and stomatal activities [106] have also been developed.Using a nanoparticle-based conducting ink, researchers printed an electrical conductometric sensor actuated by the stomatal pore,thus allowing real-time tracking of the latency of single stomata opening and closing [106].Reviews of plant nanosensors are by Kwak et al.[13] and Giraldo et al.[14].

Nanosensors can be a valuable tool for answering some basic questions in plant research.To date, our knowledge of how plant sense Na+in the environment is still rare [107,108].It is possible that Na+-specific nanosensors can be developed to monitor Na+transport in plants at high spatial and temporal resolution.The same challenge is posed by hydroxyl radicals,the most destructive ROS [95], which are also lacking a proper tool for investigating their biological roles in plants, especially under stress conditions.Current visualization methods are more based on fluorescent dyes such as hydroxyphenyl fluorescein that do not detect only hydroxyl radicals [109], impeding progress in understanding their role in plant growth and defense.Developing hydroxyl radical-specific nanosensors could allow more effective study of their functions in plants.

3.2.Making plants sensors for early stress detection

Early stress detection in crop production would help minimize agricultural losses.Although current methods of early stress detection employ either remote sensing or hyperspectral imaging to detect chlorophyll fluorescence of leaves, track plant morphological changes,or monitor plant water status,these traits reflect plant performance after stress accumulation[14].It is desirable to monitor very early stress signals.The high temporal and spatial resolution of nanosensors could allow them to complement remote sensing by monitoring stress-signaling molecules and thereby detect crop stress.In 2019,we proposed with Prof.Juan Pablo Giraldo of the University of California, Riverside, CA, USA the use of nanosensors to make plants into smart plant sensors [14].Nanosensors can recognize analytes and report this recognition via fluorescence quenching or fluorescence shifting [13,14,110].Nanosensors able to detect and respond to specific stress signaling molecules such as H2O2, glucose, and NO would allow the transduction of chemical signals to optical or radio waves for detection by agricultural equipment.Such a tool would allow more efficient farm management and early plant stress detection.Besides the non-invasive real-timein vivodetection of glucose by a QD system,recent advances in the real-timein vivodetection of H2O2in plants under stress conditions [16,111] further illustrate the potential of nano-enabled smart plants (plants engineered with nanosensors)for agriculture.Fig.2 demonstrates how nanosensors can be used for early stress detection and for engineering smart plant sensors.

However, the realization of the proposed smart plant sensor still needs more effort,especially under field conditions.Combined stresses, such as of heat and drought, commonly occur in the field[112].Combined stresses could complicate the events of chemical signaling, thus requiring higher resolution of the decoded signals generated by nanosensors in response to specific signaling molecules.Improving the sensitivity, selectivity, and accuracy of nanosensors to enable higher decoded signal resolution under field conditions would advance its application in agriculture.Efforts to build a database of the changes in chemical signaling molecules under stresses,such as those of Ca2+signatures and ROS signatures,should be added to a research agenda.Identification of new nanosensors for signaling molecules, especially those currently lacking available nanosensors, is also needed.Using zinc oxide nanoparticles as the active media in an organic field-effect transistor at room temperature permitted sensing of carbon monoxide,a plant signaling molecule [113].

4.Nano-enabled targeted delivery and controlled release of agrochemicals:Guiding the design space

4.1.Nanofertilizers and nanopesticides in agricultural production:Surface modification for controlled release and targeted delivery

Fig.2.Nanosensors can be used for early stress detection and for engineering smart plant sensors.Nanosensors have been integrated into plants to convert changes in stress signaling molecules such as H2O2, Ca2+, and NO to optical or radio waves,which were received and decoded by agricultural equipment, enabling communication between nanomaterials-engineered plants and agricultural facilities.

It has been estimated [114] that commercial fertilizers contribute to 30% to 50% of crop yield.However, some 40%-90% of agrochemicals do not reach their plant target and are lost to the environment [3].The nutrient use efficiencies of the essential N,P, and K elements by plants are 30%-35%, 18%-20%, and 35%-40%, respectively [35], leaving much scope for improving the efficacy of agrochemicals in plants.Nanopesticides and nanofertilizers are defined [35]as ‘‘a(chǎn)ny pesticide formulation or product containing engineered nanomaterials as active ingredients and having biocidal properties, either as a whole or part of the engineered structure” and ‘‘ENMs (engineered nanomaterials) that directly provide one or more required nutrients to plants” and ‘‘can also be applied to ENMs that enhance the performance, availability, or utilization of conventional fertilizers.” Nanoagrochemicals are expected to be more powerful in agricultural production than their conventional counterparts.Indeed, the estimated median gain in efficacy of nanoagrochemicals relative to conventional products is 20%-30%[41].In comparison with the bulk or MnSO4salt,foliar exposure of mung bean to Mn nanoparticles resulted in 40%-70%increases in root and shoot length, rootlet number, and biomass[115].Nanoparticles can load or encapsulate conventional agrochemicals or active ingredients to achieve controlled and targeted release of agrochemicals such as dsRNA [20], abscisic acid [116],ascorbic acid[12],and siRNA[117].More details of nanofertilizers and nanopesticides may be found in recent reviews[29,35,41,118,119].

Controlled release and targeted delivery of nanoagrochemicals to plants may be achieved by their surface modification.Among stimulus-responsive materials used to enable the controlled release of nanoformulations,pH is the most commonly(~37%studies) used stimulus [120].A GA3-HMSN/Fe3O4system [129.6 nm and about -2.0 mV for hollow mesoporous silica nanoparticles(HMSN),5.8 nm and~28.0 mV for Fe3O4;3.5 mg in 150 mL MS culture media] was built to release GA3 (gibberellic acid 3) at pH greater than 5 or pH less than 4 to promote the growth of cabbage and resulted in 44% greater stem length than a blank control[121].Zhang et al.[11] developed a temperature- and pHresponsive star nanopolymers (14-32 nm, 200 mg L-1polymer concentration) to deliver and release agrochemicals (crystal violet in this study),into tomato in response to temperature(more rapid release at 40°C than 20°C at pH 6.0).Three days after foliar application on tomato, up to 43 wt% of the star polymer PAA50-b-PNIPAm450(32 nm, 200 mg L-1polymer concentration) was translocated to other plant compartments such as roots[11].Other stimuli used include light,temperature,enzymes,and redox status.It has been proposed [122] that aptamers, oligonucleotide or peptide molecules that bind to specific targets, could be used for surface functionalization of nanoagrochemicals.Indeed, in comparison with a random peptide or a no-peptide conjugation control, highly efficient target delivery (75%) of beta cyclodextrin-conjugated quantum dots with a truncated chloroplast transit peptide into chloroplasts was achieved[12].Designing nanofertilizers or nanopesticides on the specific need and studying their uptake and fate in plants and environments could lead to their application to sustainable agricultural production.

To date, nanofertilizers and nanopesticides have not been widely adopted in agriculture, owing to biosafety questions, concerns about their environmental fate and possible transfer between species, insufficient knowledge of the possible effects on ecological systems that might result from long-term accumulation in the environment,and a delay in developing policies and regulations governing the use of nanoproducts in agriculture.More studies of the environmental fate and long-term accumulation are needed.Policies and regulations should be established to guide the use of nanoproducts in agriculture.Outreach activities aimed at farmers and the public and release of standard protocols for using nanoproducts in agricultures could lead to wider adoption of nano-enabled agriculture.

4.2.Targeting the chloroplast to tune plant performance

As mentioned above,besides offering properties such as catalytic ROS scavenging and self-fluorescence not available from conventional counterparts or bulk materials, nanomaterials can also act as platforms for controlled release and targeted delivery to specific plant organelles, organs, or tissues to reduce the waste of applied agrochemicals.Targeted delivery of nanoparticles in animal cells has been shown[123],suggesting the potential of targeted delivery of nanoproducts in plants.Indeed, recently, using quantum dots coated with beta cyclodextrin and truncated guiding peptides,researchers demonstrated the targeted delivery(with~75%delivery efficiency)of methyl viologen and ascorbic acid into chloroplasts to tune their redox status [12].This achievement opens the door not only to fine-tuning functions in cell organelles,but to reducing the use of agrochemicals.The results also show the importance of proper design of the nanoplatform:QDs guided by a random peptide with the same amino did not show improved colocalization with chloroplasts,in contrast to QDs alone.

To date,the current high efficiency of targeted delivery in plants has been achieved mostly by conjugating nanomaterials with guiding peptides [12,124], a step that requires complex synthesis and incurs high cost.Identifying new methods for the targeted delivery of nanomaterials will enable more broad applications of this technique in agricultural application.In a recent study [3], controlling nanoparticle size and charge allowed the delivery of nanoparticles to specific cell types such as epidermal and guard cells,or plant cell compartments such as the apoplast.However,this method still has the limitation of guiding the nanomaterials to specific cell organelles.In future,besides the use of guide peptides,designing nanomaterials with controlled properties such as size, charge, shape,and hydrophobicity/hydrophilicity, could allow the delivery of nanoparticles with high efficiency to plant tissues or organs,specific cell types, cell compartments, and cell organelles.Besides targeting cell organelles, developing methods to deliver nanomaterials to specific plant tissues such as the shoot apical meristem, or organs such as flowers is of interest.Positively charged CeO2nanoparticles were associated with roots and adhered primarily to roots untranslocated, while neutral and negatively charged nanoparticles were more efficiently translocated from roots to shoots than the positively charge CeO2nanoparticles[125].Efficient and targeted delivery of nanomaterials to plant tissues and organs, cells, and organelles awaits further study.And depending on the purpose of their use, the mobility of nanomaterials in plants should be designed and controlled.

A photosynthetic performance penalty is always observed in plants under abiotic stress.This process is associated with overaccumulation of ROS in plants during abiotic stress [126].Excessive accumulated ROS that overcomes the plant’s native ROS scavenging mechanism can lead to oxidative damage to protein, lipids and cell structures and even programmed cell death in plant cells[127,128].Creating plants with higher ROS scavenging ability for improved abiotic stress tolerance is an aim of plant biologists and breeders.However,genetic transformation is limited to model plants or species that can be easily transformed.A universal and scalable approach to enhance photosynthetic performance and to protect plants from abiotic stress is needed.Nanoparticles such as cerium oxide nanoparticles (nanoceria) and Mn3O4nanoparticles with ROS scavenging ability could be good candidates for use in such an approach.Indeed,plants engineered with nanoceria showed augmented ROS scavenging in leaf mesophyll cells and thus an improved carbon assimilation rate [10].Similarly, Giraldo et al.[103] showed that plants engineered with SWCNT displayed augmented ROS scavenging ability and increased electron transportation rates.Other nanomaterials with ROS scavenging ability could also help to protect chloroplasts from oxidative stress and thereby improve plant photosynthesis.With a recently developed targeted delivery approach [12], not only protecting chloroplasts from stress to improve plant photosynthesis but also turning chloroplasts into a ‘‘chloroplast factory” to allow more broad applications could become a realistic proposal.Industries producing pharmaceuticals or bioenergy and researchers interested in plant photosynthesis could benefit from this newly emerging approach.

5.Nano-enabled transgenic events:A new opportunity for plant breeding

5.1.Nanomaterials as delivery platform for transgenic events in nonmodel plant species

Currently,Agrobacterium tumefacienstransformation and gene gun bombardment are the most widely used techniques for producing transgenic events in plants.However, these two methods either are limited to a small number of genetically amenable plant species or can cause undesirable effects in plants, such as tissue damage [129,130].One of the bottlenecks of non-model species transformation is low efficiency and labor-intensive processes such as callus culture [131].In recent years, a nanobiotechnology approach showed great potential to produce transgenic events in mature wild-type plants and has the potential to be applied to a broad range of non-model plant species.Kwak et al.[21] and Demirer et al.[19] found that single-walled carbon nanotubes could deliver functional genetic materials into chloroplasts and nuclei, respectively.It is argued [132] that carbon nanotubes can enter plant cells via LEEP (Lipid Exchange Envelope Penetration)mechanisms.Variation in pH among cell compartments is regarded as the main force releasing plasmid loads from the carbon nanotubes[21].These findings hold out the prospect of using nanomaterials as platforms to deliver functional genetic materials to a broad spectrum of plant species withoutAgrobacteriuminfection or gene gun bombardment.

Recently,carbon dots[2.0-10.0nm,derivedfrom(polyethyleneimine(PEI)and thus positively charged(zeta potential not reported),40:50 as the carbon dot:siRNA mass ratio, up to 12 ng μL-1siRNA loaded] were used for siRNA delivery for gene silencing in plants,resulting in silenced GFP (Green Fluorescent Protein) expression(8 ng μL-1siRNA loaded,with almost complete loss of green fluorescence in leaves,validated by measuring a greater than 80%reduction in GFP transcript and protein levels) in both tobacco and tomato lines with constitutive expression ofeGFP(enhanced GFP) [133].Use of peptide/pDNA complexes [170 nm and ~15 mV formed at an N/P(pDNA/peptide)ratio of 1.0,1.0 μg 100 μL-1],resulted in targeted GFP expression in tobacco chloroplasts, showing that DNA molecules are translocated across cell membrane and delivered to chloroplasts by forming a complex with a chloroplast-targeting peptide[134].Here,nanomaterials provided another approach to producing transgenic events in plants, especially in non-model species.Besides acting as a delivery platform to produce transgenic events,nanomaterials can also act as a scaffold to increase the delivery efficiency of unstable chemicals or reagents such as RNA (as siRNA or dsRNA)than RNA application alone.Use of degradable,layered double hydroxide clay nanosheets(with mean diameter 45 nm,20-80 nm in the lateral dimension,0.82 nm d-value),increased the stability of the loaded dsRNA in suppressing pathogen infection on cowpea and thus in conferring virus protection for at least 20 days[20].A DNA nanostructure has been used to deliver siRNA into tobacco plants to silence constitutively expressed GFP genes[135].

Considering the relatively high cost of carbon nanotubes(which are even toxic to human beings in some cases [136]) and DNA nanostructure, researchers and farmers may prefer to use lowcost and/or biocompatible nanomaterials.More options allowing the platform to deliver functional genetic materials may lead to its wider adoption.Use PEI-coated gold nanoparticles, researchers delivered siRNA intoArabidopsisplants and silenced theNPR1(nonexpressor of pathogenesis-related gene 1) gene [137].The CDs(Carbon Dots) made with PEI could be another good candidate for delivering DNA or RNA materials [133].Theoretically, positively charged nanomaterials without heavy metals and with low cost,such as carbon dots or silica nanoparticles, could also be used as platforms for delivering negatively charged functional genetic materials into plant cells.This could be a way to reduce cost and largely remove the biosafety concerns of nanomaterials.The accessibility of the nanomaterials and the simplicity of the operation will largely determine the use and the frequency of use of this nano-enabled transgenic technique.

5.2.The possibility of designing a nanoparticle-CRISPR-Casφ complex for efficient and targeted genome editing in plants

Beside enabling transgenic events, nanomaterials have potential to act as a platform for organelle-targeted CRISPR-Cas genome editing.Though this practice is now widely adopted in plant breeding, its application is limited to specific species, genotypes,and tissues and always requires tissue culture [138,139].Nanoparticle-based delivery of the CRISPR-Cas9 system has been shown in a medical study [140].But to date, no such nanoenabled CRISPR-Cas genome editing has been reported in plants.Plant cell wall barriers(with pore sizes around 13 nm[141])could be one of the main reasons.Recently,in a huge bacteriophage,the hypercompact genome editor CRISPR-CasΦ was discovered.It is a minimal functional CRISPR-Cas system comprising a single ~ 70-k D(ca 3 nm Rmin[142])protein CasΦ and a CRISPR array[143].This system could allow researchers to better design nanoparticle-CRISPR-Cas complexes for efficient and targeted delivery to plants,such as by controlling the size of the complex to ease its passage through the plant cell wall.If so,nanoparticles could act as an efficient platform for delivering the CRISPR-Cas system to plants,even to targeted organs or organelles.Following the design of nanoparticles guided by a chloroplast transit peptide[12],the CRISPR-CasΦ system might be delivered to the chloroplast to turn it into a plant factory.Very recently,a perspective paper[144]describing the use of nanotechnology to enable CRISPR-Cas genetic engineering in plants was published in Nature Nanotechnology (following the first-round submission of the present manuscript).Readers may refer to this paper for more detail concerning the use of nanomaterials for CRISPR genome editing in cargo delivery,species independence, germline transformation and gene editing efficiency.

6.Seed nanopriming:A field requiring more mechanistic studies

For most crops,the seedling stage,especially seed germination,is sensitive or vulnerable to environmental stresses such as drought, salinity,and heat.Seed vigor improves seed germination,seedling establishment, and plant adaptation to diverse environmental conditions [145].To increase the rate and uniformity of seed germination, many techniques or approaches have been developed.They include halopriming (priming seeds with salt solution), osmopriming (priming seeds with osmotic solution at low water potential), biopriming (priming seeds with a mixture of beneficial microorganisms or bioactive molecules), solid matrix priming (priming seeds with solid material able to control the moisture content), chemopriming (priming seeds with conventional disinfectants),and thermopriming(priming seeds at various temperatures) [146].Halopriming the stress-sensitive green gram(Vigna radiata(L.) Wilczek) variety Pusa Ratna with 35 mmol L-1NaCl improved its tolerance to salinity (75 mmol L-1NaCl) and drought stress (15% PEG), resulting in a 47% (salt stress) and 28%(drought stress) increase of fresh weight relative to non-primed controls under stress [147].

Besides these priming techniques, nanoparticle-enabled seed priming (nanopriming) is an emerging approach with potential to promote crop growth and performance, especially under hostile conditions such as drought, salinity,and heat.Cotton primed with cerium oxide nanoparticles (2.1 nm, -51.7 mV, 500 mg L-1,nanopriming) showed a 41% increase in seedling fresh weight relative to a water-primed control under salt stress (200 mmol L-1NaCl) [84].Nanopriming has been reported for many crop species,including wheat (γ-Fe2O3nanoparticles [148], ZnO nanoparticles[22]),rice(silver nanoparticles[23]),sorghum(Fe2O3nanoparticles[149]), broad bean (silver nanoparticles [150]), cotton (cerium oxide nanoparticles [84]), onion (gold nanoparticles [24]), cucumber(nanoparticles of water treatment residues[151]),peanut(zinc oxide nanoparticles [152], nanoscale zerovalent iron [153]), and watermelon (Fe2O3nanoparticles [154]).Onion seed nanopriming with gold nanoparticles(93.6 nm,-8.5 mV,5.4 mg L-1,nanopriming)resulted in a 69%increase in emergence percentage and mean yield (24%) relative to an untreated control [24].Based on the increase in seed germination and the environmental impact of the embodied energy, Gilbertson et al.[34] suggested that nano-Zn/ZnO is one of the most promising seed-coating alternatives.

Although seed nanopriming shows promising results and thus high potential for agricultural use, elucidating the underlying mechanisms requires more effort.It has been proposed [84] that nanoceria priming can improve cotton salinity-stress tolerance by modulating the ROS homeostasis and ion homeostasis plant signaling pathways.Other known or proposed mechanisms of seed nanopriming are 1) reduced electrolyte leakage and increased SOD and POD activities(ZnO nanoparticles)[22],2)decreased lipid peroxidation and increased plant relative water content and photosynthetic performance (Fe2O3nanoparticles) [149], 3) lower percentages of micronuclei and chromosomal abnormalities (silver nanoparticles) [150], 4) increasing α-amylase activity to increase soluble-sugar content for supporting seedling growth and upregulation of aquaporin genes for possible greater water uptake(silver nanoparticles) [23], and 5) reducing the 12-oxophytodienoic acid level to help to break seed dormancy(Fe2O3nanoparticles) [154].

Are the mechanisms of seed nanopriming the same when one nanomaterial is applied to different plant species? Are there any mechanisms, such as modulating redox status and breaking seed dormancy, that can be commonly cited to explain seed nanopriming? Mechanisms controlling the uptake, distribution, and fate of nanoparticles and their interactions with seeds should be further investigated.How can the pore size of the seed coat affect the uptake of nanomaterials? Will the distribution of nanomaterials in seeds vary across plant species? Do the distribution patterns of nanomaterials in seeds alter its biological effect? Is this effect due to epigenetic changes or to ROS scavenging by nanomaterials?What is the critical stage or time point for the biological effects of nanomaterials during the process of seed nanopriming? Integrating seed nanopriming with seed coating technology to maintain its efficacy and function should be further investigated.Limited public information about nano-enabled seed coating technologies is available in the agrochemical industry [33].

7.Nano-enabled light harvesting and utilization:Converting nIR and UV to visible light and capturing more electrons

7.1.Using largely unused light resources:Using nanomaterials to convert nIR and UV to visible light to expand the light spectrum for plant photosynthesis

Fig.3.Using nanomaterials to expand the light spectrum and to harvest more electrons for plant photosynthesis.DCNP and UCNP nanoparticles can convert UV and nIR light into visible light.Thus, introducing these nanoparticles into plants could potentially expand the light spectrum available for photosynthesis.Nanomaterials able to capture and donate electrons might be placed in chloroplasts to increase the delivery of electrons to the electron transport chain.Both approaches have the potential to improve plant photosynthesis, especially under light-insufficient conditions.DCNP, downconversion nanoparticles; Fd, ferredoxin; FNR, ferredoxin-NADP reductase; PC, plastocyanin; PQ,plastoquinone; PSI.photosystem I, PSII, photosystem II; UCNP, upconversion nanoparticles.Solid lines represent known pathways.Dashed lines represent proposed pathways.

Plant photosynthesis is largely dependent on the availability of visible light.Most natural light sources are not well used by plants:UV light damages plants[155]and nIR light is largely not absorbed by chlorophylls in plants [156].UV-C (100-280 nm) is filtered out by the ozone layer, but UV-A (315-400 nm) and UV-B (280-315 nm)can reach the ground[155].In high-density cropping systems the leaves in the bottom tier cannot get enough visible light.Reduced visible light occurs during continuous cloudy or rainy days.New approaches that enable plants to use largely wasted light resources for photosynthesis might be useful for sustainable agriculture.

Although chlorophylldandffrom cyanobacteria was reported[157]to absorb nIR light,it could use only wavelengths up to about 750 nm.Incorporating chlorophylldandfinto higher plants might overcome the photochemical red limit.However, there would still be no way to use nIR light above 800 nm.Using nanomaterials to convert UV and nIR light into visible light usable for plant photosynthesis could be of interest to researchers, farmers, and even industry.It might afford a new opportunity to tune and augment plant photosynthesis and thus to improve crop production.Indeed,upconversion nanoparticles(UCNP)and downconversion nanoparticles (DCNP) that can convert respectively nIR and UV to visible light are widely used in biophotonics and nanomedicine [158].Yb,Nd,Er-doped UCNPs can convert nIR light (excited under 808 and 980 nm) to visible light (both 510-570 and 640-700 nm)[159].Downconversion nanoparticles such as PEI-capped β-NaYF4:Gd3+,Tb3+converted UV light at 273 nm to visible light (at about 480-630 nm) [160].Recently, when CD1:0.2 [carbon dots made with citric acid and ethanolamine (molar ratios 1:0.2);300 μg mL-1, 3.2 nm, zeta potential not reported; foliar spray]was used as a converter to convert UV radiation (300-370 nm) to PAR (Photosynthetically Active Radiation) (up to 550 nm), rice plants showed increases of 19% in shoot length, and 64% and 62%in dry weights of shoot and root relative to a control[161].Applying these UCNP and DCNP nanoparticles to plants, either by introduction into cells or spraying on the leaf surface, might help to maintain photosynthesis under visible light-insufficient conditions, such as shading or continuous cloudy days during critical stages of crop production.This nano-enabled photosynthetic light enhancement approach has potential in agriculture and related industries, such as biofuel production.Fig.3 illustrates the proposed approaches of using nanomaterials to convert nIR and UV to visible light for plant photosynthesis.

7.2.Capturing more electrons:Designing nanomaterials to improve photosynthetic efficiency

Photosynthesis fixes more than 120 billion tons of carbon in terrestrial plants [162].Light harvesting and the transferring of harvested electrons into the electron transport chain are key processes in the light reaction in plant photosynthesis.Excessive harvested electrons can lead to the formation of reactive oxygen species [163].However, during cloudy and rainy days, or under shading conditions, plants may lack harvested electrons for the light reaction.New approaches that help plants to harvest more electrons under light-insufficient conditions could improve plant photosynthesis and yield.Nanomaterials might be used to increase electron harvesting by plants.Metal organic framework nanomaterials have properties of light harvesting and energy transfer [164].Light-activated gold nanoparticles could harvest sunlight and transfer it to highly excited electrons[165].Introducing nanomaterials having light-harvesting ability into chloroplasts might improve plant photosynthesis under light-insufficient conditions.Future work should identify more light-harvesting nanomaterials and investigate their targeted and efficient delivery to chloroplasts.Fig.3 described the proposed approaches of using nanomaterials to harvest more electrons for tuning plant photosynthesis, especially under light-insufficient conditions.

8.Concluding remarks and future perspectives

We have described potential applications of plant nanobiotechnology to modern and sustainable agriculture.Plant nanobiotechnology offers prospects for 1) increasing stress tolerance, 2)stress sensing and early detection, 3) targeted delivery and controlled release of agrochemicals, 4) enabling transgenic events in non-model crop species, and 5) seed nanopriming.Nanomaterials without heavy metals and with high dispersibility should be designed for agricultural use.We argued that more studies should be conducted to investigate the biological effect of nanozymes such as Mn3O4nanoparticles in plants under stress conditions.Mechanisms underlying seed nanopriming, including the uptake,distribution, and fate of nanoparticles and their interactions with seeds, await further investigation.Plants may be engineered with nanomaterials to extend their functions, such as by turning them into chloroplast factories.Nanomaterials might be used to convert nIR and UV to visible light,and to harvest more electrons for plant photosynthesis, especially under light-insufficient conditions.Dissecting the mechanisms by which nanomaterials improve plant stress tolerance will facilitate the design of nanomaterials based on agricultural needs.Developing policies and regulatory rules could help to control biosafety risks of using nanomaterials in agriculture,and thus address public concern.We believe that nanomaterials could play an important role in agriculture.

CRediT authorship contribution statement

Honghong Wu:Conceptualization, Writing - original draft,Writing-review&editing.Zhaohu Li:Conceptualization,Writing- original draft, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the anonymous reviewers for their valuable suggestions which helped to improve the quality of this paper.We thank Prof.James Nelson from Kansas State University for polishing the English of this paper.This work was supported by the National Natural Science Foundation of China (32071971, 31901464), Fundamental Research Funds for the Central Universities(2662020ZKPY001), and the Joint Project from Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University and Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences to Honghong Wu (SZYJY2021008).